Non-typable Haemophilus influenzae (NTHi) causes diseases in humans including pneumonia, otitis media, sinusitis, and acute febrile tracheobronchitis in adults. This bacterium is also a frequent etiologic agent of otitis media in children and infants. Because infection with NTHi confers only strain-specific immunity, humans can be repeatedly infected. In fact, the most chronic disease caused by this bacterium is repeat otitis in children, resulting in treatments including repetitive exposure to antibiotics or surgery to insert drainage tubes in the ear.
None of the immunogenic compositions available to treat typable H. influenzae strains (which are characterized by a known capsule) are useful in the treatment of NTHi. Thus, considerable research has been conducted to find a component that would be useful in preventing infection caused by NTHi. In investigations of candidate components to prevent the occurrence of otitis media in humans, the bacterial lipoprotein called Protein 4 (P4; previously referred to as “outer membrane protein e”) of NTHi has been identified as a desirable component because of its ability to elicit bactericidal antibodies to the bacterium in humans. P4 has been found to elicit bactericidal antibodies, which act in synergy with antibodies against other outer membrane proteins of NTHi. Because of this, the protein may be used in conjunction with other outer membrane proteins to induce a more potent bactericidal response.
The hel gene that encodes P4 is antigenically conserved within and between strains of H. influenzae. See, e.g., U.S. Pat. Nos. 5,780,601; 5,955,580 and 5,601,831 and European Patent Nos. EP 0 606 921 and EP 0 462 210, among others, which are hereby incorporated by reference. The sequences of the hel gene (SEQ ID NO: 1) and the encoded, 274 amino acid, pre-protein (SEQ ID NO: 2) are available from the NCBI database, Accession No. M68502 and identified in Green, B. A., et al, 1991 Infect. Immun., 59(9):3191-3198. The mature P4 of NTHi is a lipidated protein of 254 amino acids in length (SEQ ID NO: 3), encoded by nucleotides 209 -970 of SEQ ID NO: 1. The 274 amino acid unprocessed pre-P4 protein has a 20 amino acid signal peptide that specifies a site for fatty acid acylation (SEQ ID NO: 2, amino acids 1-20), and is encoded by nucleotides 149-970 of SEQ ID NO: 1, with nucleotides 149-208 encoding the signal or leader peptide.
The function of the P4 protein in Haemophilus was originally thought to be hemin transport (Reidl, J., and J. J. Mekalanos. 1996 J. Exp. Med., 183:621-629.5). However, the P4 protein was later discovered to be an outer membrane enzyme, i.e., a bacterial acid phosphatase (Reilly, T. J. et al, 1999 J. Bacteriol., 181:6797-805). More recent work in the area has shown differing opinions by laboratories as to the true function of the P4 protein (Kemmer, G. et al, 2001 J. Bacteriol., 183:3974-3981; Reidl, J. et al, 2000 Mol. Microbiol., 35:1573-81; Reilly, T., and A. Smith, 1999 Prot. Exp. Purif., 17:401-409; Reilly, T. J. et al, 1999. J. Bacteriol., 181:6797-805; and Reilly, T. J. et al, 2001 FEBS Lett., 494:19-23).
The identification of P4 as an enzymatic protein has created a potential concern for its use as a component of an immunogenic composition for humans. This protein is enzymatically active and its in vivo substrate is not completely defined. While wild-type P4 has no known detrimental effects when administered into humans, it is generally accepted that non-enzymatically active proteins or those with reduced activity are preferred to enzymatically active proteins for use in immunogenic compositions to be administered to humans. An enzymatically active protein is not preferred for use as an immunogenic component because its enzymatic activity may cause an unexpected, undesired, reaction in the immunized human, other than the induction of antibodies.
Fragments of P4 protein or variant “mutant” P4 sequences described generally by the above-mentioned documents and patents are not characterized in terms of the most important property for an immunogenic composition, i.e. immunogenicity. No information is provided on the effects of mutation on immunogenicity. Further no indications are provided that changes that effect enzymatic activity have any effect on immunogenic potential. Thus, nothing in the prior art provides any direction for the selection of mutants of the P4 protein to yield a component for an immunogenic composition.
A continuing need exists in the art for therapeutic and prophylactic compositions comprising NTHi P4 variant proteins, which have reduced enzymatic activity and which are immunologically equivalent to the wild-type protein, for safe use in immunogenic compositions in humans.